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The iCycler iQ detection system for evaluating reference gene expression in normal human tissue
Umeå University, Faculty of Medicine, Radiation Sciences.
2002 In: Bio-Rad tech note, Vol. 2804Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2002. Vol. 2804
URN: urn:nbn:se:umu:diva-4260OAI: diva2:143276
Available from: 2004-11-18 Created: 2004-11-18Bibliographically approved
In thesis
1. The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
Open this publication in new window or tab >>The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor.

In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study.

In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene.

In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

61 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 932
astrocytoma, brain, EGFR, LRIG, leucine-rich repeat, real-time RT-PCR
Research subject
urn:nbn:se:umu:diva-370 (URN)91-7305-771-1 (ISBN)
Public defence
2004-12-11, Lionsalen, Onkologi, Norrlands universitetssjukhus, Umeå, 11:00 (English)
Available from: 2004-11-18 Created: 2004-11-18 Last updated: 2010-04-19Bibliographically approved

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